Method of Production of Recombinant Sucrose Synthase, Use Thereof in the Manufacture of Kits for Determination of Sucrose, Production of Adpglucose and Production of Transgenic Plants Whose Leaves and Storage Organs Accumulate High Contents of Adpglucose and Starch

ABSTRACT

Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of ADPglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of ADPglucose and starch 
     A method is described for efficient production of large quantities of soluble recombinant SS in its active form, by expression of the gene that encodes SS in a strain of  Escherichia coli . The expression vector used means that the recombinant SS produced possesses a histidine tail which facilitates its quick purification. In addition it describes sequences of mutated versions of the gene of SS that encode isoforms of SS suitable for the production of ADPG. Making use of the “wild-type” and “mutated” versions of recombinant SS, an efficient method is described for production of ADPG and UDPG. It also describes the use of SS for the production of assay kits for the determination of sucrose. Finally, it describes the production of transgenic plants which overexpress the gene of SS, either constitutively, or in leaves or storage organs, and which have a high content (both in leaves and in storage tissues) of sucrose, ADPG, G6P and starch as a result of the high ADPG-synthesizing activity of SS.

The invention relates to optimization of the production of recombinant sucrose synthase (SS) in soluble, active form employing an appropriate strain of Escherichia coli, the use of SS for making kits for determination of sucrose, design of optimized forms of SS for the synthesis of ADPglucose (ADPG), and the production of transgenic plants whose leaves and storage tissues accumulate high levels of ADPG and amylose-enriched starch as a result of overproduction of cytosolic ADPG in plants which overexpress SS.

PRIOR ART

Starch is the main storage form of carbohydrates in plants. It accumulates in large amounts in organs such as seeds (wheat, barley, maize, pea, etc.) and tubers (potato and yam among others) and is a fundamental constituent of the human diet. Furthermore, starch is widely used in the paper, cosmetic, pharmaceutical and food industries, and is also used as an essential component for the manufacture of biodegradable plastics and environment-friendly paints. Since it is made up of covalently bound glucose molecules, investigation of the processes involved in the synthesis of this polysaccharide is a top priority in various areas of industrial production.

ADPG is the universal precursor of starch biosynthesis in plants, both in heterotrophic organs (FIG. 1A) and in leaves (FIG. 2A), and it is widely assumed that its production is controlled exclusively by the enzyme ADPG pyrophosphorylase (AGPase) or ADPG synthase (EC 2.7.7.27) (Okita, T. W. (1992) Is there an alternative pathway for starch synthesis? Plant Physiol. 100, 560-56; Müller-Röber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 11, 1229-1238; Stark, D. M., Timmerman, K. P., Barry, G. F., Preiss, J., Kishore, G. M. (1992) Regulation of the amount of starch in plant tissues by ADPglucose pyrophosphorylase. Science 258, 287-282; Neuhaus, E. H., Häusler, R. E., Sonnewald, U. (2005) No time to shift the paradigm on the metabolic pathway to transitory starch in leaves. Trends Plant Sci. at press). The various applications of the starch produced in a plant are based mainly on the ratio of amylose and amylopectin, which determines the structure of the starch grain, as well as its viscosity in aqueous suspensions. This ratio of amylose and amylopectin depends on, among other things, the concentration of ADPG in the plant cell (Clarke, B. R., Denyer, K., Jenner, C. F., Smith, A. M. (1999) The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos. Planta 209, 324-329).

SS (EC 2.4.1.13, SS) (UDP-glucose:D-fructose-2-glucosyl transferase) is a reversible enzyme that catalyses the production of UDPG and fructose from sucrose and UDP. Although, as shown in FIG. 1A, SS has classically been regarded as having the role of producing UDPG, metabolic processing of which eventually gives rise to the production of starch in heterotrophic tissues such as endosperm and tubers (Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U. (1995) Evidence for the crucial role of sucrose synthase for sink strength using transgenic potato plants. Plant J. 7, 97-107; Baroja-Fernández, E., Muñoz, F. J., Saikusa, T., Rodríguez-López, M., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509; Pozueta-R{umlaut over (m)}omero, J., Muñoz, F. J., Rodríguez-López, M., Baroja-Fernández, E., Akazawa, T. (August 2003) New waves in the starch field. Lett. Plant Cell Physiol. 24-32), there are references to the potential ability of the enzyme to use other nucleotide diphosphates in vitro for the production of the corresponding sugar nucleotides (Murata, T., Sugiyama, T., Minamikawa, T., Akazawa, T. (1966) Enzymic mechanism of starch synthesis in ripening rice grains. Mechanism of the sucrose-starch conversion. Arch. Biochem. Biophys. 113, 34-44; Delmer, D. P. (1972) The purification and properties of sucrose synthase from etiolated Phaseolus aureus seedlings. J. Biol. Chem. 247, 3822-3828). Although of questionable physiological relevance (Okita, T. W. (1992) Is there an alternative pathway for starch synthesis? Plant Physiol. 100, 560-56; Müiller-Röber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 11, 1229-1238), it has been suggested that SS is capable of producing ADPG directly, which can be used for the production of starch both in heterotrophic tissues and in photosynthetic tissues (FIGS. 1B and 2B) (Pozueta-R{umlaut over (m)}omero, J., Perata, P., Akazawa, T. (1999) Sucrose-starch conversion in heterotrophic tissues of plants. Crit. Rev. Plant Sci. 18, 489-525; Baroja-Fernández, E., Muñoz, F. J., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2001) Reappraisal of the currently prevailing model of starch biosynthesis in photosynthetic tissues: a proposal involving the cytosolic production of ADPglucose by sucrose synthase and occurrence of cyclic turnover of starch in the chloroplast. Plant Cell Physiol. 42, 1311-1320; Baroja-Fernández, E., Muñoz, F. J., Saikusa, T., Rodríguez-López, M., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509; Baroja-Fernández, E., Muñoz, F. J., Zandueta-Criado, A., Morán-Zorzano, M. T., Viale, A. M., Alonso-Casajús, N., Pozueta-R{umlaut over (m)}omero, J. (2004) Most of ADPglucose linked to starch biosynthesis occurs outside the chloroplast in source leaves. Proc. Natl. Acad. Sci. USA 101, 13080-13085). According to this hypothesis (based solely and circumstantially on evidence of the biochemical type), SS is responsible for the synthesis of an important pool of ADPG molecules necessary for the biosynthesis of starch. This hypothesis has not, however, been demonstrated experimentally by genetic engineering or traditional crop improvement techniques, and is not consistent with the countless tests of the genetic and molecular type indicating that AGPase is the only source of ADPG in plants (Okita, T. W. (1992) Is there an alternative pathway for starch synthesis? Plant Physiol. 100, 560-56; Müller-Röber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 11, 1229-1238; Neuhaus, E. H., Häusler, R. E., Sonnewald, U. (2005) No time to shift the paradigm on the metabolic pathway to transitory starch in leaves. Trends Plant Sci. at press).

Sugar nucleotides such as UDPG and ADPG are produced commercially from pyrophosphorylase reactions catalysed by enzymes such as UDPG pyrophosphorylase (UGPase) and AGPase, respectively, based on the use of an expensive substance called glucose-1-phosphate (G1P). An alternative to this practice for production of sugar nucleotides is based on the use of SS, development of which has largely been hampered by the limitations of Escherichia coli for expressing and efficiently processing a large number of eukaryotic proteins. This limitation inspired some researchers to produce recombinant SS by making use of biological factories of the eukaryotic type such as yeasts (Zervosen, A., R{umlaut over (m)}omer, U., Elling, L. (1998) Application of recombinant sucrose synthase-large scale synthesis of ADP-glucose. J. Mol. Catalysis. B: Enzymatic 5, 25-28; Römer, U., Schrader, H., Günther, N., Nettelstroth, N., Frommer, W. B., Elling, L. (2004) Expression, purification and characterization of recombinant sucrose synthase I from Solanum tuberosum L. for carbohydrate engineering. J. Biotechnology 107, 135-149). Alternatively, SS intended for the production of sugar nucleotides has had to be purified by expensive processes of purification of proteins from plant extracts (patent DE4221595 (1993), Purified sucrose synthase enzyme useful for production of nucleotide-activated sugars or oligosaccharides). This SS obtained from plant extracts has the disadvantage that it has a predilection for UDP and very low affinity for ADP (Pressey R (1969) Potato sucrose synthase: purification, properties, and changes in activity associated with maturation. Plant Physiol. 44, 759-764; Nguyen-Quock, B., Krivitzky, M., Huber, S. C., Lecharny, A. (1990) Sucrose synthase in developing maize leaves. Plant Physiol. 94, 516-523; Morell, M., Copeland, L. (1985) Sucrose synthase of soybean nodules. Plant Physiol. 78, 149-154). Production of recombinant SS from cultures of E. coli has recently been achieved (Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli” Biosci. Biotech. Biochem. 61, 1500-1503; Nakai, T., Konishi, T., Zhang, Z-Q., Chollet, R., Tonouchi, N., Tsuchida, T., Yoshinaga, F., Mori, H., Sakai, F., Hayashi, T. (1997) “An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11” Plant Cell Physiol. 39, 1337-1341; Barratt, D. H. P., Barber, L., Kruger, N. J., Smith, A. M., Wang, T. L., Martin, C. (2001) Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiol. 127, 655-664; Christopher, B., William, B., Robert, H. “Bacterial sucrose synthase compositions and methods of use” Patent WO9803637). However, the production of SS in this prokaryotic system was associated with problems such as (1) the amount of SS produced was very low (30 micrograms/gram of bacteria, Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli” Biosci. Biotech. Biochem. 61, 1500-1503; Li, C. R., Zhang, X. B., Hew, C. S. (2003) “Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium goldiana. Physiol. Plantarum 118, 352-360), (2) the amount of active SS obtained was very low or zero (0.05-1.5 units/mg (Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli” Biosci. Biotech. Biochem. 61, 1500-1503; Li, C. R., Zhang, X. B., Hew, C. S. (2003) “Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium goldiana. Physiol. Plantarum 118, 352-360); 5.6 U/mg (R{umlaut over (m)}omer, U., Schrader, H., Günther, N., Nettelstroth, N., Frommer, W. B., Elling, L. (2004) Expression, purification and characterization of recombinant sucrose synthase I from Solanum tuberosum L. for carbohydrate engineering. J. Biotechnology 107, 135-149), (3) the recombinant SS had to be purified by conventional methods of purification of proteins such as chromatography, electrophoresis, isoelectric focusing, etc., which, combined, prove expensive and do not guarantee purification of the protein in a homogeneous state and (4) most of the SS is sent to inclusion bodies or is accumulated in the form of inactive aggregates as a result of the inability of the bacterium's machinery to fold the protein correctly (Miroux, B., Walker, J. E. (1996) “Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels” J. Mol. Biol. 260, 289-298).

The present invention describes the development of a system based on the use of an appropriate strain of E. coli and on the use of a suitable expression vector that permits the large-scale production and fast and easy purification of different variants of recombinant SS in its active form. Some of these variants have greater affinity for ADP than those obtained from plant extracts and can be used both for the production of UDPG and ADPG from inexpensive substances such as sucrose, UDP and ADP.

Chromatographic techniques constitute a powerful tool for determining the sucrose content of complex samples such as plant extracts, sera, urine, fruit juice, wines, fruit and foodstuffs (D'Aoust, M-A., Yelle, S, Nguyen-Quock, B. (1999) Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit. Plant Cell 11, 2407-2418; Tang, G-Q., Sturm, A. (1999) Antisense repression of sucrose synthase in carrot affects growth rather than sucrose partitioning. Plant Mol. Biol. 41, 465-479; Frias, J., Price, K. R., Fenwich, G. R., Hedley, C. L., Sorensen, H., Vidal-Valverde, C. (1996) J. Chromatogr. A 719, 213-219). Such techniques require highly specialized technical personnel and involve a large investment in equipment. Unfortunately, alternative methods based on hydrolysis of the sucrose molecule by the action of the enzyme invertase and subsequent spectrophotometric or fluorimetric determination of the molecules of glucose and/or fructose (Sweetlove, L. J., Burrell, M. M., ap Rees, T. (1996) Starch metabolism in tubers of transgenic potato with increased ADPglucose pyrophosphorylase. Biochem. J. 320, 493-498; Stitt, M., Lilley, R. M., Gerhardt, R., Heldt, H. W. (1989) Metabolite levels in specific cells and subcellular compartments of plant leaves. Methods Enzymol. 174, 518-552; Holmes, E. W. (1997) Coupled enzymatic assay for the determination of sucrose. Anal. Biochem. 244, 103-109; Methods of Analysis (1996) Code of Practice for Evaluation of Fruit and Vegetable Juices. Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Economic Community) are subject to limitations of a technical nature such as subtraction of the measurements corresponding to the endogenous glucose and/or fructose present in the sample. The abundance of glucose and/or fructose in the sample can add background noise that hampers reliable and accurate determination of sucrose. In the vast majority of cases it is necessary to carry out exhaustive controls before issuing a reliable statement on the true sucrose content of a sample (Worrell, A. C., Bruneau, J-M., Summerfelt, K., Boersig, M., Voelker, T. A. (1991) Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell 3, 1121-1130). Kits for determination of sucrose based on the use of invertase are available from companies such as Sigma, Biopharm GmbH and Megazyme. Alternatively, an automated method of sucrose determination has been developed, based on determination of the glucose-1-phosphate released by the action of sucrose phosphorylase of bacterial origin (Vinet, B., Panzini, B., Boucher, M., Massicotte, J. (1998) Automated enzymatic assay for the determination of sucrose in serum and urine and its use as a marker of gastric damage. Clin. Chem. 44, 2369-2371). The present invention describes the development of a simple, reliable and inexpensive alternative method for the determination of sucrose in a sample based on the use of SS and coupling enzymes which hydrolyse ADPG or UDPG.

Considerations concerning the factors governing the intracellular levels of ADPG have mainly revolved around regulation of the synthesizing enzyme, AGPase (Preiss, (1988) Biosynthesis of starch and its regulation. The Biochemistry of Plants. Vol. 14, Academic Press, New York, p. 182-249; Pozueta-R{umlaut over (m)}omero, J., Perata, P., Akazawa, T. (1999) Sucrose-starch conversion in heterotrophic tissues. Crit. Rev. Plant. Sci. 18, 489-525). In fact, a high proportion of the patents and scientific publications concerning the production of ADPG and the production of plants producing starches of industrial interest revolve around the use of AGPase (Stark, D. M., Timmerman, K. P., Barry, G. F., Preiss, J., Kishore, G. M. (1992) Regulation of the amount of starch in plant tissues by ADPglucose pyrophosphorylase. Science 258, 287-282; Slattery, C. J., Kavakli, H., Okita, T. W. (2000) Engineering starch for increased quantity and quality. Trends Plant Sci. 5, 291-298). However, although they are yet to be confirmed with evidence of the genetic/molecular type, recent scientific studies of a biochemical type indicate that, as shown in FIGS. 1B and 2B, SS might be involved in the direct synthesis of ADPG necessary for the biosynthesis of starch (Baroja-Fernández, E., Muñoz, F. J., Saikusa, T., Rodríguez-López, M., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509). This hypothesis is especially controversial, bearing in mind that (a) SS has never been linked to starch production in leaves, (b) presence of an ADPG translocator is required in the membranes of the plastids, connecting the cytosolic pool of the ADPG produced by SS to the starch synthase present inside the plastid and (c) the involvement of SS as an ADPG producing source is in direct conflict with many tests of the biochemical/genetic/molecular type which appear to show that AGPase is the only source of ADPG (Okita, T. W. (1992) Is there an alternative pathway for starch synthesis? Plant Physiol. 100, 560-56; Möuller-Röbber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 11, 1229-1238; Stark, D. M., Timmerman, K. P., Barry, G. F., Preiss, J., Kishore, G. M. (1992) Regulation of the amount of starch in plant tissues by ADPglucose pyrophosphorylase. Science 258, 287-282; Neuhaus, E. H., Häusler, R. E., Sonnewald, U. (2005) No time to shift the paradigm on the metabolic pathway to transitory starch in leaves. Trends Plant Sci. at press). Perhaps for all these reasons, to date plants have never been designed that overexpress SS for the production of high levels of starch. However, the present invention describes, for the first time, the production of transgenic plants that overexpress SS for increasing their production of ADPG and starch. Conversely, we show that plants that are deficient in starch as a result of absence of AGPase possess normal ADPG levels. This all shows that, as shown in FIGS. 1B and 2B, SS is involved in the direct synthesis of the ADPG required for the biosynthesis of starch and is responsible for the synthesis of most of the ADPG accumulated in the plant cell.

Although based on the approach presented in FIG. 1A, according to which SS is involved in the synthesis of UDPG (but not ADPG) in storage tissues, various works have described the production of plants with reduced content of starch as a consequence of decreased activity of SS (Chourey, P. S., Nelson, O. E. (1976) The enzymatic deficiency conditioned by the shrunken-1 mutations in maize. Biochem. Genet. 14, 1041-1055; Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U. (1995) Evidence for the crucial role of sucrose synthase for sink strength using transgenic potato plants. Plant J. 7, 97-107; Tang, G-Q., Sturm, A. (1999) Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning. Plant Mol. Biol. 41, 465-479). In this sense, there is no experimental evidence that the overexpression of SS could be used for the production of plants with high starch content as a result of the increase in levels of ADPG in accordance with the metabolic schemes shown in FIGS. 1B and 2B. However, based on the ability of SS to produce the precursor molecule of the biosynthesis of cell wall polysaccharides (UDPG), works have been published and patented which describe the production of cotton plants with high fibre content or cereals with high content of celluloses as a result of overexpression of SS (Timothy, H. J., Xiamomu, N., Kanwarpal, S. “Manipulation of sucrose synthase genes to improve stalk and grain quality” Patent WO02067662; Robert, F., Danny, L., Yong-Ling, R. “Modification of sucrose synthase gene expression in plant tissue and uses therefor” Patent WO0245485; Christopher, B., William, B., Robert, H. “Bacterial sucrose synthase compositions and methods of use” Patent WO9803637).

The invention relates firstly to the development and optimization of a method of production of large amounts of recombinant SS that is soluble, can be purified easily and has high specific activity, based on the use of a suitable strain of E. coli and on the use of an expression vector that makes it possible to obtain SS with a histidine tail. The invention further relates to the procedure followed for making kits for determination of sucrose based on the use of the enzyme product with SS activity coupled to enzymes that metabolize ADPG or UDPG. It further relates to optimization of the production of sugar nucleotides such as ADPG or UDPG starting from variants of SS specially designed for this purpose. Finally, details are given of the design of transgenic plants with high content of sucrose, ADPG and starch and a high amylose/amylopectin ratio following overexpression of SS.

DETAILED DESCRIPTION OF THE INVENTION

Amplification of a cDNA that Encodes an SS

Knowing the nucleotide sequence of wild-type sucrose synthase SS4 (Fu, H., Park, W. D. (1995) Sink- and vascular-associated sucrose synthase functions are encoded by different gene classes in potato. Plant Cell 7, 1369-1385), two specific primers were created corresponding to the 5′ and 3′ ends of the gene. Using these primers, δ 2418 base pair DNA fragment, designated SSX, from a potato-leaf cDNA library, was amplified by conventional PCR techniques. This PCR fragment was inserted in the pSK Bluescript plasmid (Stratagene), giving rise to the pSS construction (FIG. 3A), which was amplified in the host bacterium XL1 Blue.

Production of Active Recombinant SS from a Special Strain of E. Coli

pSS was digested with the NcoI and NotI restriction enzymes. The fragment released (which contains the cDNA encoding SS, SSX) was cloned on the same restriction sites of the pET-28a(+) expression plasmid (Novagen) (FIG. 3B) which possesses a nucleotide sequence in the polylinker region that encodes a histidine-rich sequence, which becomes fused with the recombinant protein. The resulting plasmid (designated pET-SS, FIG. 3C) was inserted by electroporation in various strains of E. coli. The E. coli strain BLR(DE3) (Novagen) transformed with pET-SS was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, located in the Research Building of Valencia University, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with the deposition number CECT:5850. The bacteria were incubated at 20° C. in LB medium. Overexpression of SSX was effected by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in 100 ml of cell culture grown at 20° C. After six hours of induced culture, the bacteria were collected and resuspended in 4 ml of binding buffer (Novagen, His-bind purification kits), then sonicated and centrifuged at 40,000 g for 20 minutes. The supernatant, which contains the recombinant SS with an amino acid sequence rich in histidine residues at the N-terminal end, was passed through an affinity column of the His-bind protein purification kit from Novagen. Following the instructions with the kit, SS was eluted with 6 ml of the recommended elution buffer, which contained 200 mM of imidazole instead of 1 mol. After elution, the protein was quickly submitted to dialysis to remove any trace of imidazole, which inactivates SS irreversibly.

Production of an Isoform of SS Optimized for Production of ADPG

Using suitable primers, with pSS as template, the mutated variant SS5 was designed, giving rise to the construction pSS5. This was done using the QuikChange Site-Directed Mutagenesis kit (Stratagene). pSS5 was digested with NcoI and NotI. The fragment released (which contains SS5) was cloned on the same restriction sites of the pET-28a(+) expression plasmid giving rise to pET-SS5, which was inserted by electroporation in E. coli BLR(DE3). The E. coli strain XL1 Blue transformed with pSS5 was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, located in the Research Building of Valencia University, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with the deposition number CECT:5849.

Production of Transgenic Plants that Overexpress SS4

In the present invention SS was overexpressed (a) constitutively, (b) specifically in leaves and (c) specifically in storage organs such as tubers.

For the production of plants that overexpress SS constitutively, constructions were created that were controlled by the action of the 35S constitutive promoter of the tobacco mosaic virus. Successive insertion in pSS of the 35S promoter and NOS terminator in the 5′ and 31 regions of SSX gave rise to the production of the plasmid p35S-SS-NOS, the restriction map of which is shown in FIG. 4B.

So as to be able to transfer this construction to the genome of the plants via Agrobacterium tumefaciens, it must first be cloned in a binary plasmid. For this, p35S-SS-NOS was digested successively with the enzymes NotI, T4 DNA polymerase and HindIII and was cloned within the binary plasmid pBIN20 (FIG. 4A) (Hennegan, K. P., Danna, K. J. (1998) pBIN20: An improved binary vector for Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 16, 129-131) which had previously been digested successively with the enzymes EcoRI, T4 DNA polymerase and HindIII. The plasmid thus obtained was designated pBIN35S-SS-NOS (FIG. 4C).

To overexpress SS specifically in illuminated leaves, PCR was used for amplifying the promoter region (designated RBCS) of the gene that encodes the small subunit of RUBISCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) of tobacco (Barnes, S. A., Knight, J. S., Gray, J. C. (1994) Alteration of the amount of the chloroplast phosphate translocator in transgenic tobacco affects the distribution of assimilate between starch and sugar. Plant Physiol. 106, 1123-1129). This nucleotide sequence (which confers specific expression in photosynthetically active cells) was inserted in the pGEMT-easy vector (Promega), giving rise to pGEMT-RBCSprom (FIG. 5A). This construction was digested with HindIII and NcoI and the fragment released was cloned in the corresponding restriction sites of p35S-SS-NOS, giving rise to pRBCS-SS-NOS (FIG. 5B). This construction was digested successively with HindIII, T4 DNA polymerase and NotI. The fragment released was cloned in pBIN20 digested successively with HindIII, T4 DNA polymerase and EcoRI. The resulting construction was designated pBINRBCS-SS-NOS (FIG. 5C).

After being amplified in E. coli (XL1 Blue), both pBIN35S-SS-NOS and pBINRBCS-SS-NOS were inserted in A. tumefaciens C58:GV2260 (Debleare, R., Rytebier, B., de Greve, H., Debroeck, F., Schell, J., van Montagu, M., Leemans, J. (1985) “Efficient octopine Ti plasmid-derived vectors of Agrobacterium mediated gene transfer to plants” Nucl. Acids Res. 13, 4777-4788), which was used for transforming species such as tomato (Lycopersicon sculentum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and rice by conventional techniques (Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., Fraley, R. T. (1985) “A simple and general method for transferring genes into plants” Science 277, 1229-1231; Pozueta-R{umlaut over (m)}omero, J., Houlné, G., Schantz, R., Chamarro, J. (2001) “Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation” Plant Cell Tiss. Org. Cult. 67, 173-180; Hiei, Y., Ohta, S., Komari, T., Kumashiro. T. (1994) “Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271-282). The strain of A. tumefaciens C58:GV2260 transformed with pBIN35S-SS-NOS was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, located in the Research Building of Valencia University, Burjassot Campus, Burjassot 46100 (Valencia, Spain), with the deposition number CECT:5851.

Preparation of Assay Kits for Determination of Sucrose

One of the kits designed for the determination of sucrose, shown in the following Scheme I of enzymatic reactions involved in the kit for spectrophotometric/fluorimetric determination of sucrose based on the conversion of sucrose to a sugar nucleotide and then conversion of this to glucose-1-phosphate, glucose-6-phosphate and NAD(P)H.

The kit is based on the action of SS on the sucrose molecule in the presence of a nucleotide diphosphate (e.g. UDP or ADP), releasing equimolar amounts of fructose and the corresponding sugar nucleotide. If the sugar nucleotide resulting from the reaction is UDPG, this is submitted to the action of hydrolytic enzymes of UDPG such as UDPG pyrophosphatase of the Nudix type (EC 3.6.1.45) (Yagi, T., Baroja-Fernández, E., Yamamoto, R., Muñoz, F. J., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2003) Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose. Biochem. J. 370, 409-415) or UDPG hydrolase (Burns, D. M., Beacham, I. R. (1986) Nucleotide sequence and transcriptional analysis of the E. coli ushA gene, encoding periplasmic UDP-sugar hydrolase (5′-nucleotidase): regulation of the ushA gene, and the signal sequence of its encoded protein product. Nucl. Acids Res. 14, 4325-4342). The G1P released by the action of these hydrolytic enzymes is transformed by the action of phosphoglucomutase (PGM), yielding glucose-6-phosphate (G6P), which in its turn can be made to undergo a coupling reaction with NAD(P)+ by the action of the enzyme G6P dehydrogenase (G6PDH), producing 6-phosphogluconate and NAD(P)H, which can easily be determined by fluorimetry and by spectrophotometry at 340 nm. In its turn, the NAD(P)H released can be coupled to the action of FMN-oxidoreductase/luciferase, yielding light, which is quantified spectrophotometrically.

Alternatively, as shown in scheme II, the UDPG produced can be coupled with UDPG dehydrogenase (EC 1.1.1.22) which, in the presence of NAD, gives rise to equimolar amounts of UDP-glucuronate and NADH, which can be determined by fluorimetry or by spectrophotometry at 340 nm. In its turn, the NADH released can be coupled to the action of FMN-oxidoreductase/luciferase, yielding light, which is quantified spectrophotometrically.

If the product of the reaction catalysed by the SS is ADPG, this is submitted to the action of hydrolytic enzymes of ADPG such as bacterial ADPG pyrophosphatase (EC 3.6.1.21) (Moreno-Bruna, B., Baroja-Fernández, E., Muñnoz, F. J., Bastarrica-Berasategui, A., Zandueta-Criado, A., Rodríguez-López, M., Lasa, I., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2001) Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli. Proc. Natl. Acad. Sci. USA 98, 8128-8132). The G1P released is transformed by the action of phosphoglucomutase, yielding glucose-6-phosphate (G6P), which can in turn be made to undergo a coupling reaction with NAD(P)+ by the action of the enzyme G6P dehydrogenase, producing 6-phosphogluconate and NAD(P)H, which can easily be determined by fluorimetry or spectrophotometry at 340 nm.

In any case, the schemes of enzymatic reactions coupled to the production of a sugar nucleotide mediated by SS are perfectly suitable for application to amperometric detection.

EXAMPLES OF CARRYING OUT THE INVENTION

Examples are described below, which show in detail the procedure for cloning a cDNA that encodes an isoform of SS of potato in a suitable expression vector and in a strain of E. coli optimized for the production and accumulation of the enzyme in its active form. Other examples describe the use of the recombinant SS for making assay kits for the determination of sucrose in plant samples, serum, urine, fruit juices, sweetened fruit drinks, refreshing drinks, etc. Another example describes the use of variants of SS optimized for the large-scale production of sugar nucleotides such as UDPG and ADPG. Finally, another example describes the production of plants with high content of sucrose, ADPG and starch and a high amylose/amylopectin ratio as a result of the high ADPG-producing activity in plants that overexpress SS.

EXAMPLE 1 Expression, in Escherichia coli BLR (DE3), of a Recombinant Ss with a Histidine Tail, which can be Purified Easily and has High Specific Activity

Knowing the nucleotide sequence of the SS4 gene that encodes an isoform of SS of potato, it was possible to create two specific primers whose sequences are, in the 5′-3′ direction, SEQ ID NO: 1 and SEQ ID NO: 2. Using these primers, a DNA fragment, designated as SSX, was amplified by conventional methods of PCR, from a potato tuber cDNA library, and this was inserted in a pSK Bluescript plasmid (Stratagene), which was amplified in the host bacterium XL1 Blue. The nucleotide sequence of SSX is SEQ ID NO: 3, which is slightly different from SS4 (GenBank accession number U24087). The amino acid sequence deducted from SEQ ID NO: 3 is slightly different from SS4 and is therefore designated SSX. The amino acid sequence deducted after expression of SEQ ID NO: 3 in the pET-28a(+) plasmid is SEQ ID NO: 4, which includes a histidine-rich sequence of 38 amino acids fused with the amino-terminal end of the amino acid sequence deducted from SEQ ID NO: 3.

Production of SSX in BL21(DE3) bacteria transformed with pET-SS was induced on adding 1 mM IPTG. After six additional hours of culture at 37° C., it was observed that the bacteria transformed with pET-SS accumulated a protein in aggregated form, the size of which corresponds to SS. However, these bacteria did not have SS activity. This failure in the expression of an active form of SS can be attributed to the problems that E. coli has in the correct folding of certain eukaryotic proteins of high molecular weight (Miroux, B., Walker, J. E. (1996) “Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels” J. Mol. Biol. 260, 289-298). With the aim of overcoming this problem, the capacity for production of active SS in other bacterial strains and at a temperature of 20° C. was investigated. In all of them, production of SSX was induced on adding 1 mM of IPTG. After 6 hours of additional incubation, the bacteria were sonicated and centrifuged. The resulting supernatant was analysed for SS activity. In these conditions, as shown in FIG. 6, the BLR(DE3) strain proved to be the most efficient from the standpoint of production of soluble, active SS. The E. coli strain BLR(DE3) (Novagen) transformed with pET-SS was deposited in the Spanish Type Culture Collection on 29 Oct. 2003, with the deposition number CECT:5850. The contribution of recombinant SSX in the total protein pool of CECT:5850 is approximately 20%, compared to the very low productivity of recombinant SS (30 micrograms per gram of bacteria) described in the literature (Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia Coli” Biosci. Biotech. Biochem. 61, 1500-1503; Li, C. R., Zhang, X. B., Hew, C. S. (2003) “Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium goldiana. Physiol. Plantarum 118, 352-360). The supernatant was passed through the His-Bind affinity column (Novagen), in which the recombinant protein possessing a histidine tail is retained specifically. After eluting and dialysing the purified SS, it was incubated with 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl₂/15 mM KCl/2 mM UDP. The specific activity, determined in terms of production of UDPG, was 80 units/mg of protein, much higher than the activity of 0.05-5 units/mg of recombinant SS described in the literature (Nakai, T., Tonouchi, N., Tsuchida, T., Mori, H., Sakai, F., Hayashi, T. (1997) “Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli” Biosci. Biotech. Biochem. 61, 1500-1503; Li, C. R., Zhang, X. B., Hew, C. S. (2003) “Cloning, characterization and expression analysis of a sucrose synthase gene from tropical epiphytic orchid Oncidium goldiana. Physiol. Plantarum 118, 352-360); Römer, U., Schrader, H., Günther, N., Nettelstroth, N., Frommer, W. B., Elling, L. (2004) Expression, purification and characterization of recombinant sucrose synthase I from Solanum tuberosum L. for carbohydrate engineering. J. Biotechnology 107, 135-149) and greater than 3 units/mg corresponding to the SS purified from plant extracts (Pressey R (1969) Potato sucrose synthase: purification, properties, and changes in activity associated with maturation. Plant Physiol. 44, 759-764. The unit is defined as the amount of enzyme that catalyses the production of one micromol of UDPG per minute. The affinity for UDP in the presence of 500 mM sucrose was Km(UDP)=0.25 mM, whereas the Km for sucrose was 30 mM in the presence of 1 mM UDP. This affinity for sucrose in the presence of UDP is significantly higher than that exhibited by the recombinant SS obtained in yeasts (Km=95 mM, R{umlaut over (m)}omer, U., Schrader, H., Günther, N., Nettelstroth, N., Frommer, W. B., Elling, L. (2004) Expression, purification and characterization of recombinant sucrose synthase I from Solanum tuberosum L. for carbohydrate engineering. J. Biotechnology 107, 135-149).

EXAMPLE 2 Large-Scale Production of UDPG and ADPG Based on the Use of Recombinant SS from E. Coli

Three grams of UDPG of high purity was produced efficiently and economically after incubation for 12 hours at 37° C. of 100 millilitres of a solution containing 1 M sucrose, 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl₂/15 mM KCl/100 mM UDP and 30 units of recombinant SS from potato obtained after expression of PET-SS in BLR(DE3) and subsequent purification. Reaction came to an end after heating the solution at 100° C. for 90 seconds and then centrifugation at 10,000 g for 10 minutes. The supernatant was applied to a preparative-scale HPLC chromatograph (Waters Associates) and the UDPG was purified as described in the literature (Rodríguez-López, M., Baroja-Fernández, E., Zandueta-Criado, A., Pozueta-R{umlaut over (m)}omero, J. (2000) Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis. Proc. Natl. Acad. Sci. USA 97, 8705-8710).

Production of ADPG required the generation of a mutated form of SS with an affinity for ADP much greater than that described for the SS extracted from plant tissues (Pressey R (1969) Potato sucrose synthase: purification, properties, and changes in activity associated with maturation. Plant Physiol. 44, 759-764; Nguyen-Quock, B., Krivitzky, M., Huber, S. C., Lecharny, A. (1990) Sucrose synthase in developing maize leaves. Plant Physiol. 94, 516-523; Morell, M., Copeland, L. (1985) Sucrose synthase of soybean nodules. Plant Physiol. 78, 149-154).

This isoform, designated SS5, was obtained by point mutagenesis of SSX using the QuikChange Site-Directed Mutagenesis kit (Stratagene) and successive use of the following pairs of primers whose sequences are [SEQ ID NO: 5, SEQ ID NO: 6], [SEQ ID NO: 7, SEQ ID NO: 8] and [SEQ ID NO: 9, SEQ ID NO: 10]. The nucleotide sequence obtained, designated SS5, is SEQ ID NO: 11. The changes in the amino acid sequence of SS5 (Susy 5) relative to SS4-Susy 4-(present in databases) are shown shaded in Table I. The amino acid sequence deducted after expression of SEQ ID NO: 11 in the pET-28a(+) plasmid is SEQ ID NO: 12, which includes a histidine-rich sequence of 38 amino acids fused with the amino-terminal end of the amino acid sequence deducted from SEQ ID NO: 11.

Table I includes said histidine-rich sequence of 38 amino acids fused to the amino-terminal portion of SS5.

TABLE I

The recombinant SS5 obtained after expression of pET-SS5 had a Vmax of 80 units/mg of protein and 65 units/mg of protein in the presence of UDP and ADP, respectively. The affinities for UDP and ADP in the presence of 500 mM sucrose were very similar (Km=0.2 mM both for ADP and for UDP), whereas the Km for sucrose was 30 mM and 100 mM in the presence of saturated concentrations of UDP and ADP, respectively. These kinetic parameters are very different from those described for the SS extracted from potato tuber and other organs of other species, according to which the Vmax of the enzyme is 10 times higher in the presence of UDP than in the presence of ADP (Pressey R (1969) Potato sucrose synthase: purification, properties, and changes in activity associated with maturation. Plant Physiol. 44, 759-764; Morell, M., Copeland, L. (1985) Sucrose synthase of soybean nodules. Plant Physiol. 78, 149-154; Nguyen-Quock, B., Krivitzky, M., Huber, S. C., Lecharny, A. (1990) Sucrose synthase in developing maize leaves. Plant Physiol. 94, 516-523). The E. coli strain XL1 Blue transformed with pSS5 was deposited in the Spanish Type Culture Collection, with the deposition number CECT:5849.

Three grams of ADPG of high purity was produced efficiently and economically after incubation for 12 hours at 37° C. of 100 millilitres of a solution containing 1 M sucrose, 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl₂/15 mM KCl/100 mM ADP and 30 units of recombinant SS from potato obtained after expression of pET-SS5 in BLR(DE3) and subsequent purification in a His-bind column. Reaction came to an end after heating the solution at 100° C. for 90 seconds and then centrifugation at 10,000 g for 10 minutes. The supernatant was applied to a preparative-scale HPLC chromatograph (Waters Associates) for purification of the ADPG.

EXAMPLE 3 Preparation of Enzymatic Kits for Determination of Sucrose

For determination of sucrose, the following reaction cocktails were prepared with the following components and final amounts/concentrations:

1. Kits Based on the Use of Hydrolytic Enzymes of Sugar Nucleotides:

-   -   a. 2 units of SS (recombinant or not)     -   b. 2 mM of ADP or UDP (depending on whether ADPG or UDPG is         being produced, respectively)     -   c. 2 units of ADPG pyrophosphatase or 2 units of UDPG         pyrophosphatase (depending on whether it is to be included in         the ADP or UDP reaction cocktail, respectively)     -   d. 2 units of PGM     -   e. 2 units of G6PDH     -   f. 0.5 mM of NAD(P)     -   g. reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20%         polyethylene glycol/1 mM MgCl₂/15 mM KCl     -   h. previously filtered test sample

2. Kit Based on the Use of UDPG Dehydrogenase

-   -   a. 2 units of SS (recombinant or not)     -   b. 2 mM of UDP     -   c. 2 units of UDPG dehydrogenase     -   d. 0.5 mM of NAD     -   e. reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20%         polyethylene glycol/1 mM MgCl₂/15 mM KCl     -   f. previously filtered test sample

Determination of the amount of sucrose present in the test sample is based on fluorimetric determination or spectrophotometric determination (at 340 nm) of the NAD(P)H produced according to the coupled reactions shown in schemes I and II.

For determining the sucrose content of barley seeds with different degrees of development (FIG. 7), the reactions took place in 300-microlitre wells of an ELISA plate for 3 minutes at 37° C. The volume of the test sample was 20 microlitres, and the volume of the cocktail resulting from combination of reagents a-g (kit #1) and a-e (kit #2) was 280 microlitres. The blanks contained all the components of the cocktail except SS. Measurement was carried out with a MultiSkan spectrophotometer. The values obtained, both with the kit of type “1” and with the kit of type “2” were found to be comparable to those determined using chromatographic techniques described in the introduction (Baroja-Fernández, E., Muñoz, F. J., Saikusa, T., Rodríguez-López, M., Akazawa, T., Pozueta-R{umlaut over (m)}omero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol. 44, 500-509).

EXAMPLE 4 Production of Transgenic Plants that Overexpress SS

FIGS. 8-10 present the results obtained in leaves of potato plants that overexpress SS both constitutively (35S-SS-NOS), and specifically (RBCS-SS-NOS).

As shown in FIG. 8, the SS activity in the leaves of any of these plants is 2-10 times higher than in the same organ of a wild-type plant (WT). These leaves had the following characteristics:

-   1. Clear correlation between the ADPG-producing SS activity (FIG. 8)     and levels of starch (FIG. 9) and ADPG (FIG. 10). This     characteristic was observed not only in leaves, but also in storage     tissues such as tubers and seeds (see below). -   2. High starch content (FIG. 9) relative to leaves of wild-type     plants. For example, the starch content of a leaf of a “wild-type”     potato plant grown in a photoperiod of 8 hours light/16 hours     darkness and at 20° C. is 5 micromol/gram of fresh weight, whereas a     leaf of a transgenic plant that overexpresses SS is 8 micromol/gram     fresh weight. The differences between wild-type and transgenic     plants are accentuated when the photoperiod is long, so that the     leaves of a plant that overexpresses SS contains 4 times more starch     than those of a wild-type plant. -   3. High ADPG content relative to the same tissue or organ of the     untransformed plant (FIG. 10). The average content in a leaf of a     wild-type potato plant grown in a photoperiod of 8 hours light/16     hours darkness and at 20° C. is 0.35 nanomol/gram of fresh weight,     whereas the leaves of the plants that overexpress SS can have a     content of 2.5 nanomol/gram of fresh weight. -   4. Both ADPG and starch exhibit transitory accumulation over the     photoperiod (FIG. 11). The rate of accumulation of both substances     maintains a positive correlation with the SS activity, indicating     that, contrary to what is suggested by the “classical” model of     starch biosynthesis (FIG. 2A) and confirming the hypothesis of the     “alternative” model shown in FIG. 2B, SS plays a fundamental role in     the production of ADPG and in the link between sucrose metabolism     and starch metabolism. -   5. Normal levels of soluble sugars such as glucose and fructose.     However, the levels of glucose-6-P and sucrose in transgenic leaves     are higher than those observed in the wild-type potato leaves (Table     2).

TABLE 2 Levels of metabolites (expressed in nmol/g fresh weight) in leaves of control plants (WT) and 35S- SuSy-NOS source leaves. Values significantly different from those observed in WT are shown in bold. Control 35S-SS-NOS WT 6 5 12 3 4 7 Glucose 848 ± 31   922 ± 29   850 ± 30   933 ± 29 881 ± 56 895 ± 32 871 ± 60 Fructose 996 ± 43 1,035 ± 67 1,094 ± 17 1,022 ± 10 1067 ± 58  1078 ± 63  817 ± 41 Sucrose 1,012 ± 27   1,529 ± 48 1,402 ± 68 1,642 ± 58 1,307 ± 35   1,317 ± 35   1,391 ± 70   Glucose-6-P 244 ± 28   309 ± 15   280 ± 25   271 ± 27 355 ± 23 298 ± 12  315 ± 9.8 Glucose-1-P 22.7 ± 1.9   15.5 ± 2.1   10.3 ± 1.1   9.9 ± 1.2  9.5 ± 1.5 15.2 ± 1.9 11.4 ± 1.8

-   6. The external morphology of the plants that overexpress SS is not     aberrant, when compared with that of the untransformed plants.

FIGS. 12-14 show the results obtained in potato tubers that overexpress SS constitutively (35S-SS-NOS). These results are essentially identical to those observed in tubers that overexpress SS under the control of a specific tuber promoter (promoter of the patatina gene).

As shown in FIG. 12, the SS activity in the tubers of any of these plants is ??? times greater than in the same organ of a wild-type plant. These tubers had the following characteristics:

-   1. Clear correlation between the ADPG-producing SS activity     (FIG. 12) and levels of starch (FIG. 13) and ADPG (FIG. 14). -   2. High starch content (FIG. 13) relative to tubers of untransformed     plants. For example, the starch content in the tuber of the     “wild-type” plant is approximately 300 micromol/gram of fresh weight     (equivalent to 54 mg of starch/gram of fresh weight), whereas in a     tuber that overexpresses SS it is 450-600 micromol/gram fresh     weight. -   3. High ADPG content relative to tubers of wild-type plants (FIG.     14). The average content in a wild-type tuber is 5 nanomol/gram of     fresh weight, whereas the tubers that overexpress SS can have a     content of 7-9 nanomol/gram of fresh weight.

The results obtained in rice seeds, tomato and tobacco leaves, as well as tomato fruits, are qualitatively similar to those shown in FIGS. 8-14. In all cases there was an increase in the content of starch and an increase in the amylose/amylbpectin ratio.

The production of plants with high content of ADPG and starch following overexpression of SS is a result that is totally unexpected according to the current ideas on the biosynthesis of starch (illustrated in FIGS. 1A and 2A) and perhaps explains why the design of plants that overexpress SS has not previously been adopted as a strategy for increasing starch production. The results obtained on the basis of this work suggest that SS, but not AGPase, is the fundamental source of ADPG that accumulates in plants. According to the models that are still current, AGPase is the only source of ADPG. Surprisingly, however, ADPG levels have never been investigated in AGPase-deficient plants. To explore the significance of our invention, we analysed the levels of ADPG and starch in Arabidopsis and potato plants with reduced AGPase activity for the first time. As shown in FIG. 15A, the levels of starch in AGPase-deficient TL25 Arabidopsis plants (Lin, T. P., Caspar, T., Somerville, C. R., Preiss, J. (1988) Isolation and characterization of a starchless mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity. Plant Physiol. 88, 1131-1135) are lower than those observed in the WT plants. However, the levels of ADPG are normal (FIG. 15B). In contrast, the levels of starch in AGP62 and AGP85 potato plants (Müller-Röber, B., Sonnewald, U. Willmitzer, L. (1992) Inhibition of the ADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 11, 1229-1238) are reduced relative to those observed in leaves of wild-type plants (FIG. 16A). However, the levels of ADPG are completely normal (FIG. 16B). Taken together, these observations (a) show that SS, and not AGPase, is the principal source of ADPG in plants and (b) highlight the significance of our invention after demonstrating that overexpression of SS gives rise to plants with high starch content.

DESCRIPTION OF THE DIAGRAMS

FIG. 1: Mechanisms of starch biosynthesis in heterotrophic organs. (A) “Classical” mechanism according to which SS is involved in the production of UDPG, which is eventually converted to starch after the combined action of UDPG pyrophosphorylase (UGPase), cytosolic phosphoglucomutase (PGM), plastidial phosphoglucomutase, ADPG pyrophosphorylase (AGPase) and starch synthase. (B) “Alternative” mechanism according to which SS is involved in the direct production of ADPG in the cytosol. The ADPG is then transported to the amyloplast by the action of a translocator. Once inside the amyloplast, the starch synthase utilizes the ADPG for producing starch.

FIG. 2: Mechanisms of biosynthesis of starch in leaves. (A) “Classical” mechanism according to which the entire process of starch biosynthesis takes place inside the chloroplast. According to this view, starch metabolism and sucrose are not connected. Moreover, SS does not take part in the gluconeogenic process. (B) “Alternative” mechanism of starch biosynthesis according to which SS is involved in the direct synthesis of ADPG in the cytosol. The ADPG is then transported to the interior of the plastid where the starch synthase utilizes it as substrate for the reaction of starch synthesis.

FIG. 3: Stages in construction of the pET-SS expression plasmid from pET-28a(+) and pSS.

FIG. 4: Stages in construction of the pBIN35S-SS-NOS expression plasmid from pBIN20 and p35S-SS-NOS.

FIG. 5: Stages in construction of the pRBCS-SS-NOS expression plasmid from pGEMT-RBCSprom, p35S-SS-NOS and pBIN20.

FIG. 6: Expression of pET-SS in different strains of Escherichia coli. (A) SS activity (in milliunits (mU) per milligram of bacterial protein) in bacterial extracts transformed with pET or with pET-SS. The reaction took place in the direction of degradation of sucrose and production of ADPG. The reaction cocktail contained 50 mM HEPES (pH 7.0), 1 mM EDTA, 20% polyethylene glycol, 1 mM MgCl₂, 15 mM of KCl and 2 mM of ADP. Reaction took place for 10 minutes at 37° C. (B) SDS-PAGE of protein extracts from the various strains of E. coli transformed with pET and with pET-SS. The position of the recombinant SSX is indicated with an asterisk.

FIG. 7: Determination of sucrose at different stages of development of barley endosperm using the kit based on the coupled reactions of SS, ADPG (UDPG) pyrophosphatase, PGM and G6PDH. The results were identical to those obtained in parallel by (a) use of a kit based on the coupled reactions of SS and UDPG dehydrogenase and (b) use of high-performance chromatography (HPLC) with amperometric detection in a DX-500 Dionex system connected to a Carbo-Pac PA1 column.

Abscissa: Days after flowering

Ordinate: Sucrose content (μmol/gFW)

FIG. 8: SS activity in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX following integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome. Activity is expressed in milliunits (mU) per gram of fresh weight. The unit is defined as the amount of SS required for producing one micromol of ADPG per minute.

FIG. 9: Content of starch in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX following integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome.

FIG. 10: Content of ADPG in leaves of wild-type (WT) potato plants and potato plants that overexpress SSX following integration of the constructions 35S-SS-NOS (by the action of the strain of Agrobacterium tumefaciens CECT:5851) or RBCS-SS-NOS in their genome.

FIG. 11: Transitory accumulation of (A) starch and (B) ADPG during a photoperiod of 8 hours of light and 16 hours of darkness in leaves of WT plants (), 35S-SS-NOS (▪) and RBCS-SS-NOS (▴).

FIG. 12: SS activity (referred to fresh weight, FW) in tubers of wild-type potato plants (WT), regeneration controls (RG) and potato plants that overexpress SSX (lines 4, 5, 6 and 12) after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851). The activity is expressed in milliunits (mU) per gram of fresh weight. The unit is defined as the amount of SS required for producing one micromol of ADPG per minute.

FIG. 13: Content of starch (referred to fresh weight, FW) in tubers of wild-type potato plants (WT), regeneration controls (RG) and potato plants that overexpress SSX (lines 4, 5, 6 and 12) after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851).

FIG. 14: Content of ADPG (referred to fresh weight, FW) in tubers of wild-type potato plants (WT) and potato plants that overexpress SSX after integration of the construction 35S-SS-NOS in their genome (by the action of the strain of Agrobacterium tumefaciens CECT:5851).

FIG. 15: Content of (A) starch and (B) ADPG in leaves of AGPase-deficient Arabidopsis thaliana TL25.

FIG. 16: Content of (A) starch and (B) ADPG in leaves 

1. A method of efficient production of high levels of the recombinant sucrose synthase (SS) enzyme in its soluble, active form, characterized in that it comprises: a) Obtaining, from the nucleotide sequence of the gene that encodes the isoform of SS of potato SS4, 2 primers, represented by SEQ ID NO: 1 and SEQ ID NO: 2, with which, on the basis of a potato leaf cDNA library, a cDNA fragment of 2418 base pairs represented by SEQ ID NO: 3 is amplified by PCR b) Inserting said cDNA fragment in a first vector c) Inserting said first vector in a first host microorganism where it is amplified d) Digesting the amplified construction with at least 2 restriction enzymes e) Obtaining, after prior digestion, a DNA fragment containing the cDNA that encodes SSX, the deducted amino acid sequence of which is represented by SEQ ID NO: 4 f) Cloning said fragment at the same restriction sites as a vector that contains a nucleotide sequence encoding a histidine-rich sequence, fusing said histidine-rich tail to SS, giving rise to a 2nd expression vector. g) Inserting this 2nd vector in a 2nd host microorganism where it is expressed h) Incubating said transformed microorganism in suitable culture conditions for the synthesis of SSX in its soluble, active form i) Isolating and purifying SSX in its active form
 2. The method of production of recombinant SS as claimed in claim 1, characterized in that the first expression vector used in stage b) is the plasmid pSK, which when inserted in SEQ ID NO: 3 gives rise to the construction pSS of FIG. 3A.
 3. The method of production of recombinant SS as claimed in claim 2, characterized in that the first host microorganism used in stage c) for amplifying the pSK plasmid is the E. coli bacterium XL1 Blue.
 4. The method of production of recombinant SS as claimed in claim 1, characterized in that the restriction enzymes used in stage d) are NcoI and NotI.
 5. The method of production of recombinant SS as claimed in claim 1, characterized in that, in stage f), the restriction sites where the DNA fragment released after stage d) is cloned, are the same as the plasmid pET-28a(+), shown in FIG. 3B, giving rise to the pET-SS plasmid shown in FIG. 3C.
 6. The method of production of recombinant SS as claimed in claim 1, characterized in that the 2nd host microorganism used in stage g) is the BLR(DE3) strain of Escherichia coli.
 7. The method of production of recombinant SS as claimed in claim 1, characterized in that the strain transformed in stage g) which is incubated in stage h) in suitable culture conditions for the synthesis of SSX in its soluble and active form, is CECT5850.
 8. The method of production of recombinant SS as claimed in claim 1, characterized in that the suitable culture conditions for the synthesis of SSX comprise submitting the bacterial culture to a temperature of 20° C.
 9. The method of production of recombinant SS as claimed in claim 1, characterized in that purification of SSX is preferably effected by affinity chromatography for amino acid sequences rich in histidine residues with an elution buffer that contains imidazole, preferably, at a concentration of 200 mM.
 10. The method of production of recombinant SS as claimed in claim 1, characterized in that, in order to maintain the purified SSX enzyme in its active form, said purified enzyme eluted from affinity chromatography is immediately submitted to dialysis to remove any trace of imidazole.
 11. A method of production of a recombinant potato SS5 isoform, characterized in that it comprises: a) Using the construction pSS as template and, by directed mutagenesis after successively using the pairs of primers whose sequences are [SEQ ID NO: 5, SEQ ID NO: 6], [SEQ ID NO: 7, SEQ ID NO: 8] and [SEQ ID NO: 9, SEQ ID NO: 10], obtaining a DNA fragment of 2418 base pairs represented by SEQ ID NO: 11 b) Inserting said DNA fragment in a first vector c) Inserting said first vector in a first host microorganism where it is amplified d) Digesting the amplified construction with at least 2 restriction enzymes e) Obtaining, after prior digestion, a DNA fragment that encodes SS5, the amino acid sequence of which is represented by SEQ ID NO: 12 f) Cloning said fragment at the same restriction sites as a vector that contains a nucleotide sequence encoding a His-rich sequence, fusing said His-rich tail to SS5, giving rise to a 2nd expression vector. g) Inserting this 2nd vector in a 2nd host microorganism where it is expressed h) Incubating said transformed microorganism in suitable culture conditions for the synthesis of SS5 in its soluble, active form i) Isolating and purifying SS5 in its active form
 12. The method of production of recombinant SS5 as claimed in claim 11, characterized in that stage b) gives rise to the construction pSS5.
 13. The method of production of recombinant SS5 as claimed in claim 11, characterized in that the first host microorganism used in stage c) for amplifying pSS5 is the E. coli bacterium XL1 Blue.
 14. The method of production of recombinant SS5 as claimed in claim 11, characterized in that the restriction enzymes used in stage d) are NcoI and NotI.
 15. The method of production of recombinant SS5 as claimed in claim 11, characterized in that, in stage f), the restriction sites where the DNA fragment released after stage d) is cloned, are the same as the plasmid pET-28a(+) giving rise to the plasmid pET-SS5.
 16. The method of production of recombinant SS5 as claimed in claim 11, characterized in that the 2nd host microorganism used in stage g) is the BLR(DE3) strain of Escherichia coli.
 17. The method of production of recombinant SS5 as claimed in claim 11, characterized in that the strain transformed in stage g) which is incubated in stage h) in suitable culture conditions for the synthesis of SS5 in its soluble and active form, is CECT5849.
 18. The method of production of recombinant SS5 as claimed in claim 11, characterized in that the suitable culture conditions for the synthesis of SS5 comprise submitting the bacterial culture to a temperature of 20° C.
 19. The method of production of recombinant SS5 as claimed in claim 11, characterized in that purification of SS5 is preferably effected by affinity chromatography with an elution buffer that contains imidazole, preferably, at a concentration of 200 mM.
 20. The method of production of recombinant SS5 as claimed in claim 11, characterized in that, in order to maintain the purified SS5 enzyme in its active form, said purified enzyme eluted from affinity chromatography is immediately submitted to dialysis to remove any trace of imidazole.
 21. A soluble, active recombinant SSX enzyme product, obtainable as claimed in the method of claim 1, characterized in that it has a deducted sequence represented by SEQ ID NO: 4 and displays sucrose synthase (SS) activity.
 22. The soluble, active recombinant SSX enzyme product as claimed in claim 21, characterized in that it has a specific activity of 80 U/mg protein in the presence of sucrose and UDP, and kinetic parameters of Km(UDP)=0.25 mM and Km(sucrose)=30 mM.
 23. A SS5 isoform of the soluble, active recombinant enzyme product of claim 21, characterized in that it has a deducted amino acid sequence shown in SEQ ID NO: 12 and displays sucrose synthase (SS) activity.
 24. The recombinant SS5 isoform as claimed in claim 23, characterized in that it has a specific activity of 80 U/mg protein and 60 U/mg protein in the presence of UDP and ADP, respectively, and kinetic parameters with respect to UDP and ADP of Km(UDP)=Km(ADP)=0.3 mM.
 25. A method for use of the enzyme product of claim 21 in the production of UDPG, characterized by incubation of UDP and SSX in suitable conditions followed by isolation and purification of the UDPG produced.
 26. The method as claimed in claim 25, characterized in that it comprises: a) Incubating 100 ml of the following solution for 12 h at 37° C.: Sucrose 1 M HEPES, pH 7.0 50 mM EDTA 1 mM Polyethylene glycol 20% MgCl₂ 1 mM Kcl 15 mM UDP 100 mM SSX 30 U

b) Stopping the reaction by heating, preferably at 100° C. for 90 s c) Centrifuging at 10000 g for 10 min d) Chromatographing the supernatant by HPLC, eluting and purifying the UDPG by conventional methods.
 27. A method for use of the enzyme product of claim 23 in the production of ADPG, characterized by incubation of ADP and SS5 in suitable conditions, followed by isolation and purification of the ADPG produced.
 28. The method as claimed in claim 27, characterized in that it comprises: e) Incubating 100 ml of the following solution for 12 h at 37° C.: Sucrose 1 M HEPES, pH 7.0 50 mM EDTA 1 mM Polyethylene glycol 20% MgCl₂ 1 mM KCl 15 mM ADP 100 mM

f) Stopping the reaction by heating, preferably at 100° C. for 90 s g) Centrifuging at 10000 g for 10 min h) Chromatographing the supernatant by HPLC, eluting and purifying the ADPG by conventional methods.
 29. A method comprising use of an enzyme product with sucrose synthase (SS) activity in the manufacture of assay kits for the spectrophotometric/fluorimetric/amperometric determination of sucrose.
 30. The method as claimed in claim 29, characterized in that it comprises the following incubation medium: a. 2 units of SS b. 2 mM of ADP c. 2 units of ADPG pyrophosphatase of plant, animal or microbial origin d. 2 units of PGM e. 2 units of G6PDH f. 0.5 mM of NAD(P) g. 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl2/15 mM KCl h. previously filtered test sample.
 31. The method as claimed in claim 29, characterized in that it comprises the following incubation medium: a. 2 units of SS b. 2 mM of UDP c. 2 units of UDPG pyrophosphatase of plant, animal or microbial origin d. 2 units of PGM e. 2 units of G6PDH f. 0.5 mM of NAD(P) g. 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl2/15 mM KCl h. previously filtered test sample.
 32. The method as claimed in claim 29, characterized in that it comprises the following incubation medium: a. 2 units of SS b. 2 mM of UDP c. 2 units of UDPG dehydrogenase d. 0.5 mM of NAD e. 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl2/15 mM KC1 f. previously filtered test sample.
 33. The method as claimed in claim 29, characterized in that the SS present in the assay kit is, indiscriminately, SS4, SS5 or SSX or a combination thereof.
 34. A method comprising use of DNA that encodes SS, in the production of transgenic plants that express SS, characterized by inserting a genetic construction that contains and expresses said DNA in a suitable vector and transferring said genetic construction to the genome of a plant.
 35. The method as claimed in claim 34, characterized in that the cDNA used encodes SSX.
 36. The method as claimed in claim 35, characterized in that it comprises the following steps: a) Successive insertion, in the pSS plasmid, of the promoter 35S and of the terminator NOS in the 5′ and 3′ regions, respectively, of the SSX gene or any other version that encodes SS, to produce the plasmid p35S-SS-NOS, the restriction map of which is shown in FIG. 4B b) Successive digestion of p35S-SS-NOS with the enzymes NotI, T4 DNA polymerase and HindIII c) Cloning of the fragment produced, within the binary plasmid pBIN20, previously digested successively with EcoRI, T4 DNA polymerase and HindIII, obtaining the plasmid pBIN35S-SS-NOS shown in FIG. 4C d) Amplification of pBIN35A-SS-NOS in E. coli (XL1 Blue) e) Insertion of the genetic construction amplified in the preceding stage in Agrobacterium tumefaciens C58:GV2260, obtaining a transformed strain CECT 5851 f) Transfection of plants with the transformed strain CECT
 5851. 37. The method as claimed in claim 35, characterized in that it comprises the following steps: a) Successive insertion, in the pGEMT plasmid, of the promoter of the gene that encodes the small subunit of RUBISCO for producing the plasmid pGEMT-RBCSprom, the restriction map of which is shown in FIG. 5A b) Digestion of pGEMP-RBCS with the enzymes HindIII and NcoI for inserting the fragment released at the corresponding restriction sites of p35S-SS-NOS, giving rise to pRBCS-SS-NOS, the restriction map of which is shown in FIG. 5B. c) Successive digestion of pRBCS-SS-NOS with HindIII, T4 DNA polymerase and NotI and cloning of the fragment released in pBIN20 digested successively with HindIII, T4 DNA polymerase and EcoRI, giving rise to pBINRBCS-SS-NOS, the restriction map of which is shown in FIG. 5C. d) Amplification of pBINRBCS-SS-NOS in E. coli (XL1 Blue) e) Insertion of the genetic construction amplified in the preceding stage in Agrobacterium tumefaciens C58:GV2260 and transfection of plants with the transformed strain.
 38. Transgenic plants obtainable by the method of use of claim 34, characterized by overexpression of an SS enzyme activity.
 39. The transgenic plants as claimed in claim 38, characterized in that said overexpression assumes levels of SS enzyme activity of the order of 2-10 times greater than those present in the same tissue of a non-transgenic wild-type plant.
 40. The transgenic plants as claimed in claim 38, characterized in that they are preferably selected from tobacco, potato, tomato or rice plants.
 41. The transgenic plants as claimed in claim 38, characterized in that in addition they have higher contents of sucrose, G6P, ADPG and starch, than those observed in the same tissue or organ of the corresponding wild-type plants, grown in identical conditions.
 42. The transgenic plants as claimed in claim 40, the leaves of which have a content of sucrose, G6P, ADPG and starch, and an amylose/amylopectin ratio, higher than those observed in the leaves of the corresponding wild-type plants.
 43. The transgenic plants as claimed in claim 40, whose roots, tubers and/or seeds have a content of sucrose, G6P, ADPG and starch, and an amylose/amylopectin ratio, higher than those observed in the same tissues or organs of the corresponding wild-type plants. 